1. Field of the Invention
This invention relates to hypertension assessment and a method to screen hypertension drugs. In particular, the present invention relates to assessing hypertension based on arachidonic acid-derived metabolites. The present invention relates to measurement of gucuronidated DHET and glucuronidated DHET metabolites to monitor hypertension and development of drugs for arachidonic acid epoxygenase-, epoxide hydrolase- and UDP-glucuronosyl transferase-dependent hypertension.
2. Description of Related Art
Arachidonic acid (“AA”) is a component of cellular membranes and plays a critical role as a mediator of cell and organ function through its metabolic cascade. The AA cascade includes prostaglandin synthases, lipoxygenases, and cytochromes P450 (CYPs). The CYP pathway is composed of lipoxygenases-like (allylic oxidation), ω/ ω-1 oxygenases and epoxygenases (olefin epoxidation), which metabolize AA to produce 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs), 16- to 20-hydroxyeicosatetraenoic acids (OH-AAs), and 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs), respectively (1). Epoxide hydrolases hydrolyze biologically active EETs to their corresponding dihydroxyeicosatrienoic acids (DHETs) and UDP-glucuronosyl transferase transforms DHETs to glucuronidated DHETs.
CYPs which have been identified to be AA epoxygenases are CYP2C11, CYP2C23, CYP2B1, and CYP2B2 expressed in rats (1), CYP2C1 and CYP2C2 expressed in rabbits (2,3), and CYP2C8 and CYP2C9/2C10 expressed in human (4). CYP2J is abundantly expressed in extrahepatic tissues (5-7).
EETs have potent vasoactive properties (vasodilator or vasoconstrictor). Urinary excretion of Na+, EETs and DHETs decreased after inhibition of AA epoxygenase activity by treating rats with clotrimazole, which induced salt-sensitive and clotrimazole-dependent hypertension. A salt-sensitive phenotype of the Dahl rat was associated with a lack of increases in renal AA epoxygenases after intake of a high salt diet (8,9).
A spontaneous hypertensive rat (SHR) study indicated that ω/ω-1 hydroxylase activity of kidney microsomes was significantly higher than that of normotensive Witstar Kyoto rats (WKY) whereas AA epoxygenase activity (EETs+DHETs) showed no difference between two strains at any age group tested (10). Thus, it was generally known that the developmental phase of hypertension was linked to increases in the activity of kidney microsomal ω/ω-1 hydroxylase. Indeed, recently the gene coding for CYP4A2 (ω/ω-1 hydroxylase) was found to be preferentially expressed in SHR (10,11).
However, the result obtained at the inventor's laboratory revealed that DHET levels in urine specimens obtained from SHR was ˜56-fold higher than those of WKY (U.S. Pat. No. 6,534,282). The inventor also found that 14,15-DHET exists in the urine as free (ethyl acetate extractable) and conjugated (ethyl acetate unextractable) forms and antibodies produced against 14,15-DHETs recognize both free and several conjugated forms. Cross-reactivity of the antibodies with the conjugated form of 14,15- and 8,9-DHETs was further demonstrated by ELISAs using 14,15- and 8,9-DHETs conjugated to bovine serum albumin. Epoxide hydrolase activity may be measured by the ELISAs against 14,15-DHET. Anti-DHET does not recognize 14,15-EET (U.S. Pat. No. 6,534,282).
The specificity of the antibody developed against 14,15-DHET was further investigated utilizing slot blot analysis. The 14,15-DHET conjugated BSA, BSA alone and 8,9-DHET conjugated to BSA were blotted onto cellulose membrane. Slot blot analysis was carried out with anti-14,15-DHET. Though the same amount of protein is loaded to each lane (proteins were visualized by amido black staining), the antibody cross-reacted with 14,15-DHET conjugated BSA whereas the antibody failed to cross-react with 8,9-DHET which is structurally very similar to 14,15-DHET (U.S. Pat. No. 6,534,282).
Surprisingly, it is now noted that glucuronidated DHETs in a biological system can function as a biomarker for hypertension caused by increased activities of epoxide hydrolase- and UDP-glucuronosyl transferase. The AA metabolite-dependent hypertension drugs can be developed utilizing the glucuronidated marker.